Orthomyxoviruses (influenza virus) segmented negative strand RNA viruses, constitute an important group of human and animal pathogen. The long term goal of our project is to elucidate the processes involved in the replication of standard as well as defective interfering (DI) RNAs. In addition, we want to delineate the steps involved in the amplification of DI RNAs and DI-mediated interference of standard virus replication. During the previous project periods, we have shown that influenza DI particles contain, in addition to standard RNA segments, small subgenomic RNAs (DI RNAS) which are responsible for DI-mediated interference. These DI RNAs arise predominantly, if not exclusively, from the three polymerase (P) genes (PBl, PB2 PA) by one or more internal deletions. These DI RNAS; therefore, retain both 5' and 3' sequences of the progenitor genes and are transcriptionally active both in vitro and in infected cells. In addition, some of these transcripts are produce defective proteins. The objective of the present project is to answer a number of questions using DI RNA as a marker, we want to determine if the assembly and packaging of influenza virus eight RNA segments occur randomly or in a selective fashion. This would answer a long standing puzzle of RNAs (RNPs) into infectious particles. Since influenza replication and transcription take place in the cellular nuclei, we would like to define the determinants required for transporting these P proteins into the nuclei. Furthermore, since polymerase protein function as 3P RNP complex, we would like to define the steps and requirements of 3P complex formation and finally, use the 3P complex in reconstituting an in vitro transcription replication system. These experiments would enable us to define the function of individual P proteins and the locatio of critical domain(s) and site(s) in the RNA and proteins involved in transcription and replication.